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Metabolic engineering of plant volatiles
Natalia Dudareva1 and Eran Pichersky2
Metabolic engineering of the volatile spectrum offers enormous
potential for plant improvement because of the great
contribution of volatile secondary metabolites to reproduction,
defense and food quality. Recent advances in the identification
of the genes and enzymes responsible for the biosynthesis of
volatile compounds have made this metabolic engineering
highly feasible. Notable successes have been reported in
enhancing plant defenses and improving scent and aroma
quality of flowers and fruits. These studies have also revealed
challenges and limitations which will be likely surmounted as
our understanding of plant volatile network improves.
Addresses
1
Department of Horticulture and Landscape Architecture, Purdue
University, West Lafayette, IN 47907, United States
2
Department of Molecular, Cellular and Developmental Biology,
University of Michigan, 830 North University Street, Ann Arbor, MI
48109, United States
Corresponding author: Dudareva, Natalia ([email protected])
Current Opinion in Biotechnology 2008, 19:1–9
This review comes from a themed issue on
Plant Biotechnology
Edited by Joe Chappell and Erich Grotewold
0958-1669/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2008.02.011
Introduction
Plants produce an amazing diversity of low molecular
weight organic compounds known as secondary or
specialized metabolites [1]. More than 1% of these
metabolites are lipophilic molecules with low boiling
points and high vapor pressures at ambient temperature.
They are mainly represented by terpenoids, phenylpropanoids/benzenoids, fatty acid derivatives and amino acid
derivatives. These volatile compounds are released from
leaves, flowers and fruits into the atmosphere and from
roots into the soil. The primary functions of airborne
volatiles are to defend plants against herbivores and
pathogens, to attract pollinators, seed dispersers, and
other beneficial animals and microorganisms, and to serve
as signals in plant–plant interaction. The contribution of
volatiles to plant survival and overall reproductive success
in natural ecosystems, and their impact on agronomic and
other commercial traits, including yield and food quality,
suggest that modification of volatile production via
genetic engineering has the potential to improve cultivated plant species.
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Metabolic engineering requires a basic understanding of
the biochemical pathways and the identification of the
genes and enzymes involved in the synthesis of volatile
compounds. In the last decade a renewed interest in these
questions combined with technical advances have led to
both a large increase in the number of plant volatiles
identified as well as remarkable progress in discovering
the genes and enzymes of volatile biosynthesis. Numerous attempts have been made to modulate volatile profiles in plants via metabolic engineering to enhance direct
and indirect plant defense and to improve scent and
aroma quality of flowers and fruits [2–5]. While a few
projects have been successful in achieving the desired
goals, many other attempts have resulted in meager
enhancement of volatiles or in other unpredicted metabolic consequences such as further metabolism of the
intended end products or deleterious effects on plant
growth and development. In this review we highlight
the latest advances in plant volatile research and discuss
recent efforts to modify volatile traits, emphasizing the
challenges and limitations of metabolic engineering of
volatile profiles.
Improvement of plant defense via metabolic
engineering
In the past two decades it has been well documented that
in response to herbivore attack plants emit diverse
volatile blends that may be composed of more than
200 different compounds [6]. These emitted volatiles
can directly intoxicate, repel or deter herbivorous insects
[7–12], or they may attract natural predators and parasitoids of the offending herbivores, thus indirectly protecting the signaling plant from further damage (e.g.,
tritrophic interactions) [13–15]. The growing number of
reports on the involvement of volatiles in plant defense
suggests that plant protection in agricultural and forest
ecosystems can be enhanced via the modulation of the
volatile spectrum by metabolic engineering, thereby providing an alternative pest-management strategy based on
biological control [16]. However, several requirements
must be fulfilled for successful improvement of plant
defense [4]. First, the herbivore enemies capable of sufficiently controlling the herbivore population must be present in the locality where the crop is grown. Second, the
introduced or enhanced plant volatile blends must provide
key attractants for herbivore enemies, and volatile release
must be synchronized with herbivore activity. Finally, the
released volatiles should not increase the attractiveness of
the plant to non-specific herbivores [17].
In addition to direct and indirect induced defenses,
herbivore-induced volatiles can act as airborne signals
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COBIOT-516; NO OF PAGES 9
2 Plant Biotechnology
that warn neighboring plants about the pathogen attack
and prime them to respond more strongly against future
insect attack [18,19,20] or serve as signals within a
plant and prime systemic defenses [21,22,23]. Although
the molecular mechanisms underlying priming are
unknown, priming prepares the plant or its undamaged
parts for accelerated defense but delays the response until
the actual herbivore attack. Since the defense network
remains mostly dormant until actual herbivore attack
priming-related costs are substantially lower than those
of the induced direct defense, and the benefits of priming
outweigh its costs when disease occurs [24]. Priming
crops by planting a few transgenic plants that constantly
emit defense volatiles in the field (Figure 1) may offer an
efficient form of plant protection and provide an
advantage to non-transgenic receiver plants. However,
a full use of this approach will only become possible with a
comprehensive understanding of the molecular mechanisms of volatile-induced priming, the determination of
the major volatile signal components that trigger it and
their species specificity, and the identification of reliable
molecular markers for the primed state.
Although it is likely that the array of volatiles emitted
from a given species evolved as adaptations to specific
challenges, it is not surprising that certain plant enemies
have in turn evolved to take advantage of such emissions
to locate their plant ‘prey’ for food, egg deposition, and
the raising of their young. In addition, it was recently
shown that volatiles released from plants can also provide
chemical cues to parasitic weeds, the most damaging
agricultural pests, for host location and discrimination
[25]. An understanding of the parasite–host interactions
and, in particular, the role of volatiles in these plant–plant
interactions, will aid in the development of new tactics for
the non-herbicidal control of weed populations via the
metabolic alteration of the hosts’ volatile spectrum.
Although to date very little is known about the attractive
and repelling properties of specific plant volatiles, the key
compounds involved in plant–insect and plant–plant
interactions and the molecular mechanisms of their
action, the modulation of the volatile spectrum has
already been proven to be a useful strategy for enhancing
volatile-based plant defenses. The overexpression of
strawberry linalool/nerolidol synthase (FaNES1) targeted
to chloroplasts resulted in transgenic Arabidopsis producing high levels of linalool which repelled the aphid Mysus
persicae in dual-choice assays [11]. The ectopic expression
of the same FaNES1 gene in transgenic potato increased
Figure 1
Priming with plant beacons. A few transgenic plants engineered for continuous synthesis and emission of airborne signals can ‘prime’ a field of nontransgenics and thereby increase their ability to resist attacking herbivores and pests.
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Metabolic engineering of plant volatiles Dudareva and Pichersky 3
the level of linalool and affected tritrophic interactions,
creating transgenic plants that were more attractive to
predatory mites than the uninfested wild-type plants
[2,26]. Improvement of Arabidopsis indirect defense
was also achieved by overexpressing FaNES1 in mitochondria, which contains the sesquiterpene precursor
farnesyl diphosphate (FPP). This manipulation led to
the synthesis and emission of (3S)-(E)-nerolidol as well
as the C11 homoterpene 4,8-dimethyl-1,3(E),7-nonatriene [(E)-DMNT], believed to be a degradation product of nerolidol, and rendered the plants attractive to the
carnivorous predatory mites Phytoseiulus persimilis, natural
enemies of spider mites [27].
In another successful example, the overexpression of a
maize terpene synthase gene (TPS10) in Arabidopsis
resulted in transgenic plants with strong emission of
several sesquiterpenes that are typically released (in
maize) after herbivory by lepidopteran larvae. These
transgenic plants were more attractive to the female
parasitic wasp Cotesia marginiventris, which had had a
previous oviposition experience with larvae of the potential host [28]. Moreover, production of the volatile patchoulol and 13 additional sesquiterpene products in
transgenic tobacco overexpressing patchoulol synthase
(PTS) deterred tobacco hornworms, a majority of which
had migrated away from leaves of the transgenic plants to
the leaves of wild-type plants and consumed 20–50%
more of the wild-type plants within six hours [29].
Attempts at metabolic engineering of volatile signals
involved in direct and indirect defenses have not been
restricted to terpenoids. An increase in (Z)-3-hexenal, a
major green leaf volatile, was achieved in transgenic
tobacco plants overexpressing either the yeast acyl-CoA
D9 desaturase or the insect acyl-CoA D11 desaturase. The
expression of these transgenes resulted in elevated levels
of 16:1 fatty acids and increased 13-lipoxygenase activity,
which catalyzes the first step to hexenal production from
a-linolenic acid [30]. While the effect of elevated levels of
(Z)-3-hexenal on insect behavior was not investigated in
this study, the negative effect of this compound on aphid
performance was demonstrated in transgenic potato
plants with reduced levels of the hydroperoxide lyase
enzyme, which is responsible for the cleavage of fatty acid
hydroperoxides to C6 aldehydes [10].
These studies have demonstrated both the potential of
genetic engineering for the improvement of plant defense
as well as the role of some volatile compounds in plant–
insect interactions. Despite this progress, they have also
revealed the effect of genetic perturbations on plant
growth and development, and uncovered some challenges to achieving efficient production of the desired
volatile terpenoid compounds. In fact, the diversion of
carbon to linalool production in Arabisopsis via FaNES1
overexpression, while not effecting the levels of plastidwww.sciencedirect.com
derived isoprenoids such as chlorophylls, lutein and bcarotene, led to a growth-retardation phenotype that was
inherited through several generations of transgenic plants
[11]. Emission of linalool in transgenic potato resulted in a
more severe phenotype; in addition to growth retardation,
plants had bleached leaves after their transfer from in vitro
to the greenhouse [26]. Leaf chlorosis, vein clearing, and
reduced stature were also observed in transgenic tobacco
producing high levels of patchoulol as a result of the
expression of PTS coupled with FPP synthase, both
targeted to the plastids [29]. These observed phenotypes could be the consequences of the depletion of
isoprenoid precursors for other metabolites essential for
plant growth and development, or possibly the toxic
effects of the newly introduced terpenoids in plant cells.
For successful metabolic engineering of the volatile spectrum, it is important to produce and emit sufficient
amounts of the desired compounds. However, the metabolic fate of newly synthesized compounds will be determined by the entire biochemical repertoire of the plant
used. Since a complete understanding of the biochemical
repertoire in any plant species is not available, it is
difficult to predict how much of the desired compound
will actually remain in the desired form. For example, the
newly synthesized compounds may be affected by
enzymes that are normally present in the cell and have
broad substrate specificity, such as dehydrogenases, glucosyl transferases, and others [31]. Presently there is little
knowledge of such enzymes in general and their specific
distribution in different plant species. In fact, in transgenic Arabidopsis constitutively expressing FaNES1, part
of the free linalool was subjected to hydroxylation and
glycosylation by endogenous enzymes (and, as mentioned
previously, when nerolidol was produced, some of it was
degraded to the C11 homoterpene (E)-DMNT). Moreover, the total levels of glycosides of linalool and its
derivatives were at least 10-fold higher than those of
the free alcohols [11]. Interestingly, this glycosylation
profile was different from that detected in linalool-producing transgenic potato, while the 8-hydroxy derivatives of
linalool ((E)-8-hydroxy linalool, (Z)-8-hydroxy linalool
and (E)-8-hydroxy 6,7-dihydrolinalool) were identical
in both species [11,26].
The initial attempts to increase terpenoid production in
transgenic plants showed that metabolic engineering of
sesquiterpenes is a more challenging task and is not as
straightforward as the generation of monoterpenes, which
are formed exclusively or at least predominantly via the
methylerythritol phosphate (MEP) pathway in the plastids. In many cases, FPP, which is expected to be produced in relatively large amounts for sterol biosynthesis,
is not readily available for catalysis by introduced sesquiterpene synthases (reviewed in [5]). In addition, the
contribution of the cytosolic mevalonic acid (MVA) and
plastidic MEP pathways to sesquiterpene formation and
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4 Plant Biotechnology
thus trafficking of isoprenoid intermediates between
organelles depends on the plant species, tissue and physiological state of the plant [32–35]. To date, the overexpression of TPS10 and PTS in Arabidopsis and tobacco,
respectively, represent the two most successful attempts
at producing high levels of volatile sesquiterpenes by
enzymes targeted to the cytosol [28,29]. However, to
achieve emission of (3S)-(E)-nerolidol and (E)-DMNT in
Arabidopsis, FaNES1 had to be directed to the mitochondria [27]. In addition, targeting PTS along with FPP
synthase to the plastids increased the amount of produced
patchoulol up to 100 times compared with its cytosolic
formation [29].
It has only recently been appreciated that plants emit
volatile compounds from their roots into the rhizosphere
[36–38]. Such volatiles may help the plant attract
beneficial microorganisms and ward off harmful ones.
They may also be useful in competition between plant
species [39]. However, it has been shown that some
parasitic plants use belowground volatile compounds to
locate their hosts [38]. At present there are no reports of
genetic engineering attempts to change root volatile
emission in order to improve plant fitness; this is clearly
a very fertile area for future work.
Metabolic engineering of floral volatiles
In contrast to metabolic engineering of vegetative volatiles where the effect of altered emission profiles on insect
behavior was investigated, the impact of changes in floral
scent on insect attraction has not yet been studied. Moreover, perception assessments have generally been limited
to sensory evaluations by humans, whose odor threshold
perception is much lower than that of insects [40,41]. In
such experiments, metabolic engineering of floral volatiles was considered successful when the changes in scent
profiles were significant enough for human detection. For
example, the olfactorily detectable enhancement of volatiles emitted from flowers and leaves was achieved in
transgenic tobacco via the introduction of three citrus
monoterpene synthases [42,43]. In another experiment,
the redirection of the metabolic flux from the anthocyanin
pathway towards benzoic acid in transgenic carnations
resulted in an increase of methylbenzoate production
which was sufficient for olfactory detection by humans
[44]. However, many more attempts to modify the scent
bouquet were less successful for different reasons including the absence of suitable substrates for the introduced reaction [45,46], modification of the scent
compound into a non-volatile form [47], insufficient
levels of emitted volatiles for olfactory detection by
humans, or masking of introduced compound(s) by other
volatiles [48].
The elimination of some volatile compounds from the
floral bouquet is another approach which has recently
been used for scent modifications. Transgenic petunias
Current Opinion in Biotechnology 2008, 19:1–9
lacking methylbenzoate [49], phenylacetaldehyde [50],
benzylbenzoate and phenylethylbenzoate [51], and isoeugenol [52] were obtained via RNAi-mediated posttranscriptional gene silencing. The effect of these changes on
human perception has not yet been tested with the
exception of the plants with lower levels of methylbenzoate emission. In this case, the panelists reacted negatively
by complaining that flowers were less fragrant [49].
Improvement of aroma quality of fruits,
vegetables and herbs
Volatiles are important determinants of the overall aroma
properties and taste of fruits [53]. In nature, volatiles
contribute to seed dispersion by increasing fruit attractiveness. Volatiles released from vegetative parts of plants
may also be attractive to some animals or insects as
foodstuff, even though they are unpalatable to most other
herbivores.
The presence of volatiles in fruits, vegetables and herbs has
important influence on the cultivation of many plant
species. As extensive breeding programs are undertaken
to maximize certain attributes of foodstuff – for example,
overall yield (i.e. size), total solids, sugar content, or pigmentation – less attention is devoted to enhancing or even
maintaining volatile production. As a result many current
cultivars of domesticated plant species produce less volatiles than their wild relatives or earlier cultivars [54].
Reintroduction of aroma volatiles can be achieved by
classical breeding, as was done in tomato by crossing it
with its relative L. peruvianum [55]. However, this is a
laborious and time-consuming process which requires the
monitoring of a complex trait. For example, volatile
collections and analyses must initially be done with
expensive gas chromatography–mass spectrometry
(GC–MS) instruments and subsequently human evaluations must also be performed by subjective test panels as
it is not yet possible to predict how humans will react to a
given mixture of volatile compounds. Human evaluation
of smells is particularly subjective because of interspecific
variation in the ability to detect specific compounds and
the lack of shared vocabulary to describe specific smells.
These complications have indeed contributed to the lack
of emphasis in most breeding programs on the aroma of
produce.
Genetic engineering can ameliorate some drawbacks of
classical plant breeding and enhance aroma of fruits. One
advantage of this approach is that it is less complex –
introducing a single trait at a time. Another is that it allows
the introduction of genes whose coding information may
not be present in the cultivar. Several recent reviews have
enumerated general problems and pitfalls of genetic
engineering for biochemical traits (e.g. [56,57]), therefore
we will not repeat these caveats here, with the exception
of two worth mentioning again. First is that the addition
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Table 1
Genes used in the metabolic engineering of [volatile] compounds
AADC, aromatic L-amino acid decarboxylase; AAT, alcohol acetyltransferases; ADH, alcohol dehydrogenase; AOS, allene oxide synthase; BPBT,
benzylalcohol/phenylethanol benzoyltransferase; BSMT, benzoic acid/salicylic acid carboxyl methyltransferase; CCD, carotenoid cleavage dioxygenase; CFAT, coniferyl alcohol acetyltransferase; GLV, green leaf volatiles; HPL, hydroperoxide lyase; ODO1, ODORANT1; PAAS, phenylacetaldehyde synthase; PAR, 2-phenylacetaldehyde reductase; LOX, lipoxygenase.
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6 Plant Biotechnology
of a single gene is unlikely to result in a substantial
production of the desired volatile if the formation of this
compound is the end result of a long metabolic pathway.
Second, and perhaps even more important, is that a single
new volatile is generally unlikely to change the consumers’ overall perception regarding the flavor and aroma
quality of produce.
The overexpression of a yeast D9-desaturase [58] and a
non-specific alcohol dehydrogenease (ADH) [59,60] in
tomato fruit were early attempts to circumvent these
problems. In these cases, the concentrations of various
aroma compounds derived from fatty acids such as (Z)-3hexenol, (Z)-3-hexenal and/or the ratios of the aldehydes
to alcohols changed. The higher levels of alcohols in
transgenic fruits were associated with more intense ripe
flavor by taste panelists [59]. However, neither of these
manipulations introduced new aroma compounds.
The introduction of the Clarkia breweri linalool synthase
(LIS) gene into tomato under the control of the fruitspecific E8 promoter was the first attempt at adding a new
compound to fruit flavor. It resulted in the accumulation
in the fruit of small amounts of linalool and its oxidation
product, 8-hydroxylinalool, which were detectable by
both GC–MS and the human nose [61]. This metabolic
manipulation was accomplished because linalool is produced from geranyl diphosphate (GPP) by LIS in a single
step and GPP is an intermediate in the synthesis of
carotenoids, a pathway that is highly active in ripening
tomato fruits. The synthesis of linalool and 8-hydroxylinalool did not affect the total amounts of carotenoids
produced by the fruit; however, the amounts of these
monoterpenes were also not sufficient to substantially
change the overall flavor perception by humans (Lewinsohn and Pichersky, unpublished data).
A much stronger effect on flavor perception was recently
achieved by Davidovich-Rikanati et al. [62] by expressing geraniol synthase (GES) in tomato under the polygalacturonase promoter, another fruit ripening-specific
promoter. Geraniol is also an acyclic monoterpene alcohol
that is synthesized in one step from GPP. Unlike linalool,
which is a tertiary alcohol whose hydroxyl group cannot
be further oxidized, geraniol is a primary alcohol that can
easily be oxidized to geranial by non-specific alcohol
dehydrogenases [62]. GES-transgenic tomato fruits synthesized large amounts of geraniol, which led to a noticeable decrease in pigmentation. Moreover, transgenic
fruits further metabolized geraniol to geranial, which
underwent spontaneous tautomerization to neral. Neral
and geranial together make a mixture called citral, which
imparts a strong lemon flavor. Geranial and neral were
also further metabolized to geranic and neric acids,
respectively. Additional modifications of geranial and
neral resulted in the formation of nerol, citronellol, citronellal, citronelic acid, citronellyl acetate, and rose oxide
Current Opinion in Biotechnology 2008, 19:1–9
[62]. When these transgenic fruits were evaluated by a
test panel of 34 people, the majority of participants (80%)
indicated that the fruits had stronger aroma, and more
than 60% of the panel members preferred the transgenic
fruits over the non-transgenic ones.
Attempts to modify vegetative plant volatile production
for human consumption have lagged behind the efforts
made with tomato fruit. However, recent work in peppermint on boosting the production of terpenes favored
by humans (e.g. menthol) and decreasing the synthesis of
unfavored compounds (e.g. menthofuran) were partially
successful [63,64,65]. Using antisense technology, transgenic plants were obtained that had a 50% reduction in
menthofuran concentration, while other transgenic plants
showed some increase in the concentrations of limonene,
a cyclic monoterpene. However, an assessment of consumer responses to the aroma properties of these transgenic mint plants has not yet been reported.
Conclusions
In the past several years we have witnessed significant
progress in both identifying genes and enzymes involved
in the biosynthesis of volatiles compounds and our ability
to manipulate the volatile spectrum in plants (Table 1).
However, metabolic manipulations often yield unpredictable results, highlighting our lack of a comprehensive
understanding of plant metabolic networks and their
regulation, including our rudimentary knowledge concerning network organization, the subcellular localization
of the enzyme involved, competing pathways, metabolic
channeling, flux-controlling steps and possible feedback
control. Additional molecular and biochemical characterization in combination with metabolic flux analysis and
computer assisted modeling [51] must be carried out to
provide the theoretical foundation for successful manipulation of the volatile spectrum and to identify targets for
future metabolic engineering. The identification of key
compounds involved in volatile-induced plant defenses,
as well as insect attraction, and their effects on insect
behavior in field studies, will also greatly contribute to
target selection.
Acknowledgements
This work is supported by National Science Foundation /USDA-NRI
Interagency Metabolic Engineering Program, grants MCB 0331333 (ND)
and MCB 0331353 (EP), and by grant from Fred Gloeckner Foundation, Inc
to ND. This paper is contribution No 2007-18268 from Purdue University
Agricultural Experimental Station.
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